1. Field of the Invention
The present invention relates to a fatty acid hydropcroxide lyase protein, which has activity for 9-hydroperoxide substrates and which is present in muskmelon (Cucumis melo), and the gene encoding the protein. The present invention also relates to the means for expressing the hydroperoxide lyase and methods of using the lyase in the field of organic synthesis.
2. Background Art
Plants produce various volatile compounds that give rise to the characteristic flavors and odors of the particular plant. Unsaturated fatty acids like linoleic and linolenic acids are precursors of flavor compounds such as n-hexanal, hexan-1-ol, 2(E)-hexen-1-al, 2(E)-hexen-1-ol, 3(Z)- hexen-1-al, 3(Z)- hexen-1-ol (also known as pipol), 3-(Z)-nonenal, (3Z,6Z)-nonadienal, 3-(Z)-nonenol, (3Z,6Z)-nonadienol, 2-(E)-nonenal, (2E,6Z)-nonadienal, 2-(E)-nonenol, and (2E,6Z)-nonadienol. These compounds are used widely in flavors, particularly fruit flavors, and are used by the aroma industry for a fruit aroma. The demand for these flavor compounds has grown to exceed their supply from traditional sources, thus motivating research efforts toward finding alternative natural ways of obtaining these materials.
The synthesis of these flavor compounds starts from free (polyunsaturated) fatty acids such as linoleic (9(Z), 12(Z)-octadecadienoic) and .alpha.-linolenic (9(Z), 12(Z), 15(Z)- octadecatrienoic) acids. In nature, these acids are released from cell membranes by lipolytic enzymes after cell damage. Fatty acid hydroperoxides are formed by the action of a lipoxygenase (LOX) and are subsequently cleaved by a hydroperoxide lyase to give C.sub.6 - and C.sub.9 -volatile flavor compounds together with .omega.-oxoacids. The cleavage of 13-hydroperoxides yields C.sub.6 -compounds, including hexanal and (3Z)-hexenal, and the cleavage of 9-hydroperoxides yields C.sub.9 -compounds, (3Z)-nonenal and (3Z,6Z)-nonadienal. In the presence of isomerases, these aldehydes are isomerized to (2E)-enals. Furthennore, alcohol dehydrogenases can convert the aldehydes into their corresponding alcohols.
The HPL enzymes have proven difficult to study because they are membrane bound and are present in only small quantities in plant tissue. The HPL enzymes have been characterized as 13-HPLs or 9-HPLs, according to their substrate specificity. The 13-HPL enzyme was identified for the first time in banana fruits (Tress1 and Drawert, 1973) and was subsequently studied in a number of different plant materials, including watermelon seedlings (Vick and Zimmerman, 1976), apple and tomato fruits (Schreier and Lorenz, 1982), tomato leaves (Fauconnier et al., 1997), cucumber seedlings (Matsui, et al, 1989), and soybean seedlings (Olias et al., 1990). The 13-HPL enzyme has been purified from tea leaves (Matsui et al., 1991) and, more recently, from green bell pepper fruits (Shibata et al., 1995), tomato leaves (Fauconnier et al., 1997), sunflower (Itoh and Vick, 1999), guava (PCT application, WO 9958648 A2), and banana (European Patent Application, Publication No. EP 0801133 A2). A 9-hydroperoxide specific HPL has been identified in pear (Kim and Grosch, 1981). There have been studies that suggested the presence of a third type of HPL that cleaves both 9- and 13-hydroperoxides. (Matsui et al. 1989; Hornostaj and Robinson, 1998).
Crude sources of lyases are currently used in an industrial process for the production of flavors and aromas. (See, e.g., U.S. Pat. No. 5,464,761). In this process, a solution of the required substrates made from linoleic or linolenic acid (obtained from sunflower and linseed oils, respectively) using freshly prepared soybean flour as a source of LOX. This solution is then mixed with a freshly prepared puree of whole fruit, as the crude source of HPL. The aldehyde products are then isolated by distillation. When the alcohols are required, fresh baker's yeast is added to the hydroperoxide solution before it is mixed with the fruit puree. This yeast contains an active alcohol dehydrogenase enzyme that reduces the aldehydes as they are formed by the HPL.
There are a number of disadvantages to this industrial process. The principal disadvantage is the requirement of large quantities of fresh fruit. Such a requirement means that the process has to be operated in a country where fresh fruit is cheaply and freely available. Even when such a site is found, availability is limited to the growing season of the fruit.
A second disadvantage is that the desired enzyme activities are rather dilute in the sources employed. This means that relatively large amounts of soy flour, fruit puree, and yeast have to be used in the process. The large volumes of these crude materials that are required for industrial production place physical constraints on the yields of flavor and aroma compounds that can be achieved.
A third disadvantage is that it is a large-volume batch process, which, by its nature, does not make maximum use of the HPL's catalytic activity, is relatively labor intensive, and generates a large amount of residual organic material. The residual organic material must subsequently be transported to a compost farm or otherwise discarded.
The present invention overcomes these limitations and disadvantages related to the source of muskmelon 9-HPL by providing purified and recombinant muskmelon 9-HPL proteins, nucleic acids, expression systems, and methods of use thereof.